Bacterial infections remain among the most common and deadly causes of human disease. Infectious diseases are the third leading cause of death in the United States and the leading cause of death worldwide (Binder et al. (1999) Science 284, 1311-1313). Multi-drug-resistant bacteria now cause infections that pose a grave and growing threat to public health. It has been shown that bacterial pathogens can acquire resistance to first-line and even second-line antibiotics (Stuart B. Levy, The Challenge of Antibiotic Resistance, in Scientific American, 46-53 (March, 1998); Walsh, C. (2000) Nature 406, 775-781; Schluger, N. (2000) Int. J. Tuberculosis Lung Disease 4, S71-S75; Raviglione et al., (2001) Ann. NY Acad. Sci. 953, 88-97). New approaches to drug development are necessary to combat the ever-increasing number of antibiotic-resistant pathogens.
The present invention provides one such approach.
RNA polymerase (RNAP) is the molecular machine responsible for transcription and is the target, directly or indirectly, of most regulation of gene expression (Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr. Opin. Structl. Biol. 11, 155-162; Cramer, P. (2002) Curr. Opin. Structl. Biol. 12, 89-97; Murakami & Darst (2003) Curr. Opin. Structl. Biol. 13, 31-39; Borukhov & Nudler (2003) Curr. Opin. Microbiol. 6, 93-100; Landick, R. (2001) Cell 105, 567-570; Korzheva & Mustaev (2001) Curr. Opin. Microbiol. 4, 119-125; Armache, et al. (2005) Curr. Opin. Structl. Biol. 15, 197-203; Woychik & Hampsey (2002); Cell 108, 453-463; Asturias, F. (2004) Curr. Opin. Genet Dev. 14, 121-129; Cramer, P. (2004) Curr. Opin. Genet. Dev. 14, 218-226; Geiduschek & Kassayetis (2001) J. Mol. Biol. 310, 1-26). Bacterial RNAP core enzyme has a molecular mass of ˜380,000 Da and consists of one β′ subunit, one β subunit, two α subunits, and one ω subunit; bacterial RNAP holoenzyme has a molecular mass of ˜450,000 Da and consists of bacterial RNAP core enzyme in complex with the transcription initiation factor σ (Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr. Opin. Structl. Biol. 11, 155-162; Cramer, P. (2002) Curr. Opin. Structl. Biol. 12, 89-97; Murakami & Darst (2003) Curr. Opin. Structl. Biol. 13, 31-39; Borukhov & Nudler (2003) Curr. Opin. Microbiol. 6, 93-100). Bacterial RNAP core subunit sequences are conserved across Gram-positive and Gram-negative bacterial species (Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr. Opin. Structl. Biol. 11, 155-162; Iyer, et al. (2004) Gene 335, 73-88). Eukaryotic RNAP I, RNAP II, and RNAP III contain counterparts of all bacterial RNAP core subunits, but eukaryotic-subunit sequences and bacterial-subunit sequences exhibit only limited conservation (Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr. Opin. Structl. Biol. 11, 155-162; Cramer, P. (2002) Curr. Opin. Structl. Biol. 12, 89-97).
Bacterial RNAP is a proven target for antibacterial therapy (Chopra, et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) Trends Biochem. Sci. 29, 159-162). The suitability of bacterial RNAP as a target for antibacterial therapy follows from the fact that bacterial RNAP is an essential enzyme (permitting efficacy), the fact that bacterial RNAP subunit sequences are conserved (providing a basis for broad-spectrum activity), and the fact that bacterial RNAP subunit sequences are only weakly conserved in eukaryotic RNAP I, RNAP II, and RNAP III (providing a basis for therapeutic selectivity).
The rifamycin antibacterial agents—notably rifampicin, rifapentine, and rifabutin—function by binding to and inhibiting bacterial RNAP (Chopra, et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Floss & Yu (2005) Chem. Rev. 105, 621-632; Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005) Cell 122, 351-363). The rifamycins bind to a site on bacterial RNAP adjacent to the RNAP active center and sterically and/or allosterically prevent extension of RNA chains beyond a length of 2-3 nt (Chopra, et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Floss & Yu (2005) Chem. Rev. 105, 621-632; Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005) Cell 122, 351-363). The rifamycins are in current clinical use in treatment of Gram-positive and Gram-negative bacterial infections (Chopra, et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Floss & Yu (2005) Chem. Rev. 105, 621-632; Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005) Cell 122, 351-363). The rifamycins are of particular importance in treatment of tuberculosis; the rifamycins are first-line anti-tuberculosis agents and are the only anti-tuberculosis agents able rapidly to clear infection and prevent relapse (Mitchison, D. (2000) Int. J. Tuberc. Lung Dis. 4, 796-806). The rifamycins also are of importance in treatment of bacterial infections relevant to biowarfare or bioterrorism; combination therapy with ciprofloxacin, clindamycin, and rifampicin was successful in treatment of inhalational anthrax following the 2001 anthrax attacks (Mayer, et al. (2001) JAMA 286, 2549-2553), and combination therapy with ciprofloxacin and rifampicin, or doxycycline with rifampicin, is recommended for treatment of future cases of inhalational anthrax (Centers for Disease Control and Prevention (2001) JAMA 286, 2226-2232).
The clinical utility of the rifamycin antibacterial agents is threatened by the existence of bacterial strains resistant to rifamycins (Chopra, et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Floss & Yu (2005) Chem. Rev. 105, 621-632; Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005) Cell 122, 351-363). Resistance to rifamycins typically involves substitution of residues in or immediately adjacent to the rifamycin binding site on bacterial RNAP—i.e., substitutions that directly decrease binding or function of rifamycins. A significant and increasing percentage of cases of tuberculosis are resistant to rifampicin (1.4% of new cases, 8.7% of previously treated cases, and 100% of cases designated multidrug-resistant, in 1999-2002; Schluger, N. (2000) Int. J. Tuberc. Lung Dis. 4, S71-S75; Raviglione, et al. (2001) Ann. N.Y. Acad. Sci. 953, 88-97; Zumia, et al. (2001) Lancet Infect. Dis. 1, 199-202; Dye, et al. (2002) J. Infect. Dis. 185, 1197-1202; WHO/IUATLD (2003) Anti-tuberculosis drug resistance in the world: third global report (WHO, Geneva)). Strains of bacterial bioweapons agents resistant to rifampicin can be, and have been, constructed (Lebedeva, et al. (1991) Antibiot. Khimioter. 36, 19-22; Pomerantsev, et al. (1993) Antibiot. Khimioter. 38, 34-38; Volger, et al. (2002) Antimicrob. Agents Chemother. 46, 511-513; Marianelli, et al. (204) J. Clin. Microbiol. 42, 5439-5443).
In view of the public-health threat posed by rifamycin-resistant and multidrug-resistant bacterial infections, there is an urgent need for new classes of antibacterial agents that (i) target bacterial RNAP (and thus have the same biochemical effects as rifamycins), but that (ii) target sites within bacterial RNAP distinct from the rifamycin binding site (and thus do not show cross-resistance with rifamycins). (See Chopra, et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S (2004) Trends Biochem. Sci. 29, 159-162.)
Recently, crystallographic structures have been determined for bacterial RNAP and eukaryotic RNAP II (Zhang et al., (1999) Cell 98, 811-824; Cramer et al., (2000) Science 288, 640-649; Naryshkin et al., (2000) Cell 101, 601-611; Kim et al., (2000) Science 288, 1418-1421; Korzheva et al., (2000) Science 289, 619-625; Ebright, R. (2000) J. Mol. Biol. 304, 687-689; Cramer et al., (2001) Science 292, 1863-1876; Gnatt et al., (2001) Science 292, 1876-1882; Mekler et al., (2002) Cell 108, 599-614; Murakami et al., (2002) Science 296, 1280-1284; Murakami et al., (2002) Science 296, 1285-1290; Vassylyev et al., (2002) Nature 417, 712-719; Bushnell et al., (2004) Science 303, 983-988; Westover et al., (2004) Science 303, 1014-1016; Armache, et al., (2003) Proc. Natl. Acad. Sci. USA 100, 6964-6968). Moreover, cryo-EM structures have been determined for bacterial RNAP and eukaryotic RNAP I (Opalka, et al. (2000) Proc. Natl. Acad. Sci. USA 97, 617-622; Darst, et al. (2002) Proc. Natl. Acad. Sci. USA 99, 4296-4301; DeCarlo, et al. (2003) J. Mol. Biol. 329, 891-902).
Structures also have been determined for RNAP complexes with nucleic acids, nucleotides and inhibitors (Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005) Cell 122, 351-363; Campbell, et al. (2005) EMBO J. 24, 674-682; Artsimovitch, et al. (2004) Cell 117, 299-310; Tuske, et al. (2005) Cell 122, 541-522; Temiaov, et al. (2005) Mol. Cell. 19, 655-666; Vassulyev, et al. (2005) Nature Structl. Biol. 12, 1086-1093; Gnatt, et al. (2001) Science 292, 1876-1882; Westover, et al. (2004a) Science 303, 1014-1016; Westover, et al. (2004b) Cell 119, 481-489; Ketenberger, et al. (2004) Mol. Cell. 16, 955-965; Bushnell, et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 1218-1222; Kettenberger, et al. (2005) Natl. Structl. Mol. Biol. 13, 44-48).
The structures reveal that RNAP-bacterial or eukaryotic—has a shape reminiscent of a crab claw. The two “pincers” of the “claw” define the active-center cleft that can accommodate a double-stranded nucleic acid—and which has the active-center Mg2+ at its base (FIG. 1A). The largest submit (β′ in bacterial RNAP) makes up one pincer, termed the “clamp,” and part of the base of the active-center cleft. The second-largest subunit (β in bacterial RNAP) makes up the other pincer and part of the base of the active-center cleft.
The structures further reveal that the RNAP clamp can exist in a range of distinct conformational states—from a fully open clamp conformation that permits unimpeded entry and exit of DNA to a fully closed clamp conformation that prevents entry and exit of DNA (FIG. 1A; Ebright (2000) J. Mol. Biol. 304, 687-698; Darst (2001) Curr. Opin. Structl. Biol. 11, 155-162; Cramer (2002) Curr. Opin. Structl. Biol. 12, 89-97; Murakami & Darst (2003) Curr. Opin. Structl. Biol. 13, 31-39; Borukhob & Nudler (2003) Curr. Opin. Microbiol. 6, 93-100; and Landick (2001) Cell 105, 567-570). It has been proposed that the clamp opens to permit DNA to enter the active-center cleft in transcription initiation, closes after DNA enters the active-center cleft in transcription initiation, and further closes, or acquires further stability in the closed state, in transcription elongation. Clamp closure is proposed to be responsible for the high stability of initiation complexes and for the exceptionally high stability and exceptionally high processivity of elongation complexes.
The “switch region” is located at the base of the clamp (FIG. 1A). The switch region serves as a hinge that permits rotation of the β′ “pincer” relative to the remainder of RNAP, and, correspondingly, permits opening or closing of the RNAPactive-center cleft. The switch region adopts different conformations when the β′ pincer is rotated out of the active-center cleft (open-clamp state, required for entry of DNA into active-center cleft) and when the β′ is rotated into the active-center cleft (closed-clamp state, required for stable binding of DNA within active-center cleft) (FIG. 1B; Cramer (2002) Curr. Opin. Structl. Biol. 12, 89-97; Landick (2001) Cell 105, 567-570; Cramer, et al. (2001) Science 292, 1863-1876; Gnatt, et al. (2001) Science 292, 1876-1882).
Several residues of the switch region make direct contacts with DNA phosphates in the transcription elongation complex (Gnatt, et al. (2001) Science 292, 1876-1882; Westover, et al. (2004a) Science 303, 1014-1016; Westover, et al. (2004a) Science 303, 1014-1016; Westover, et al. (2004b) Cell 119, 481-489; Kettenberger, et al. (2004) Mol. Cell. 16, 955-965). Furthermore, it has been proposed that direct contents between the switch region and DNA phosphates might coordinate, and even might mechanically couple, clamp closure and DNA binding (Cramer, et al. (2002) Curr. Opin. Structl. Biol. 12 89-97; Landick, et al. (2001) Cell 105, 567-570; Cramer, et al. (2001) Science 292, 1863-1876; and Gnatt, et al. (2001) Science 292, 1876-1882).